Radioimmunoassay is an in vitro assay technique introduced in 1960 by Berson and Yalow to measure hormone levels in blood plasma. It uses the principles of a competitive binding reaction and measurement of radioactivity. In the assay, a radiolabeled antigen competes with unlabeled antigen from a test sample to bind to antibodies. The amount of radiolabeled antigen bound is inversely proportional to the concentration of unlabeled antigen in the sample. This sensitive and specific technique can detect antigen or antibody levels and has applications in endocrinology, oncology, toxicology and other areas of medical testing.

1. RADIOIMMUNOASSAY
BY:
FARHA BANU,
MSC MICROBIOLOGY

2. HISTORY
• The technique was
introduced in 1960
by Berson and Yalow
as an assay for the
concentration of
insulin in plasma.
• It represented the
first time that
hormone levels in
the blood could be
detected by an
invitro assay.
Dr. Rosalyn Yalow became the first
female to win a Nobel Prize with
her work on the radioimmunoassay

3. ANTIGENS
• An antigen is a substance with the ability to
induce an immunological response. They
typically enter the body from an infection. They
are recognized at their epitopes by B cells or
by the T cell receptor on T cells.

4. ANTIBODIES
• Antibodies are produced by the
B-lymphocytes. They are glycoproteins
belonging to the “immunoglobulin supergene
family” that are produced in response to a
foreign substance in the body.
• Antibodies have a generally common structure,
but have regions that vary among them to
accommodate the specific antigens.

5. LABELS IN RIA
• Immunoassays require the use of labeled materials in order
to measure the amount of antigen or antibody present. A
label is a molecule that will react as part of the assay, and
in doing so produce a signal that can be measured in the
solution. Examples of a label include a radioactive
compound, or an enzyme that causes a change of color in
a solution or its fluorescence .

6. INTRODUCTON
• Radio immune assay (RIA): As the name
indicates, it is an immunological assay to
analyze any antigen or anti-body in the patient’s
serum to diagnose the disease.
• This is one of the most sensitive & specific
methods of immune assays available.
• It involves competitive binding of radio-labeled
antigen and unlabeled antigen to a high-affinity
antibody.
• The sensitivity range is 0.0006–0.006 µg
antibody/ml.

7. PRINCIPLE
• It involves three principles which make it most
specific & sensitive than other immune assays.
1. An immune reaction i.e. antigen, antibody binding.
2. A competitive binding or competitive displacement
reaction. (It gives specificity)
3. Measurement of radio emission. (It gives
sensitivity)

8. PRINCIPLE
1. IMMUNE REACTION:
• When a foreign biological substance enters into
body blood stream through non oral route, body
recognizes the specific chemistry on surface of
foreign substance as antigen and produces specific
antibodies against the antigen so as nullify the
effects and keep the body safe. The antibodies are
produced by body immune system so, it is an
immune reaction.

9. PRINCIPLE
2. A competitive binding or competitive displacement
reaction:
• This is a phenomenon wherein when there are two antigens
which can bind to same antibody, the antigen with more
concentration binds extensively with the limited antibody
displacing other. So here in the experiment, radiolabelled
antigen is allowed to bind to high affinity antibody. Then
when patient serum is added unlabelled antigens in it start
binding to the antibody displacing the labeled antigen.

11. PRINCIPLE
3.Measurement of radio emission:
• Once the incubation is over, then washings
are done to remove any unbound antigens.
Then radio emission of the antigen antibody
complex is taken, the gamma rays from radio
labeled antigen are measured.

12. APPROCH FOR RIA
• The first step to set up an RIA is to determine the amount of
antibody needed to bind 50–70% of a fixed quantity of
radioactive antigen (Ag*) in the assay mixture. This ratio of
antibody to Ag* is chosen to ensure that the number of
epitopes presented by the labeled antigen always exceeds
the total number of antibody binding sites. Consequently,
unlabeled antigen (from patient serum) added to the
sample mixture will compete with radiolabeled antigen to
bind to the limited number of antibody.

13. • Even a small amount of unlabeled antigen added to the
assay mixture of labeled antigen and antibody will cause a
decrease in the amount of radioactive antigen bound, and
this decrease will be proportional to the amount of unlabeled
antigen added.
• To determine the amount of labeled bound antigen, the Ag-
Ab complex is precipitated to separate it from free antigen
(antigen not bound to Ab), and the radioactivity in the
precipitate is measured. A standard curve can be generated
using unlabeled antigen samples of known concentration (in
place of the test sample), and from this plot the amount of
antigen in the test mixture may be precisely determined.

14. PROCEDURE

15. PROCEDURE
• The labeled antigen is mixed with antibody at
a concentration that saturates the antigen-
binding sites of the antibody.
• Then test samples of unlabeled antigen of
unknown concentration are added in
progressively larger amounts.

16. PROCEDURE
• The antibody does not distinguish labeled from
unlabeled antigen, so the two kinds of antigen
compete for available binding sites on the antibody.
As the concentration of unlabeled antigen
increases, more labeled antigen will be displaced
from the binding sites.
• The decrease in the amount of radiolabeled antigen
bound to specific antibody in the presence of the
test sample is measured in order to determine the
amount of antigen present in the test sample.

17. PROCEDURE
• The antigen is generally labeled with a
gamma-emitting isotope such as I125, but
beta-emitting isotopes such as tritium (3H)
are also routinely used as labels.
• The radiolabeled antigen is part of the assay
mixture; the test sample may be a complex
mixture, such as serum or other body fluids,
that contains the unlabeled antigen.

19. ADVANTAGES & DISADVANTAGES
ADVANTAGES
• Highly specific: Immune reactions are specific.
• High sensitivity:Immune reactions are sensitive.
DISADVANTAGES
• Radiation hazards.
• Requires specially trained persons.
• Lab requires special license to handle radioactive
material.
• Storage and disposal of radioactive material.

20. APPLICATIONS
• Detection of narcotic drugs.
• Measurement of growth hormone levels,
immunoglobulins.
• Allergy diagnosis.
• Detect drug abuse or drug poisoning.
• Early cancer detection and diagnosis.
• Blood bank screening for the hepatitis virus.

21. THANK YOU